Methods for making and delivering Rho-antagonist tissue adhesive formulations to the injured mammalian central and peripheral nervous systems and uses thereof

ABSTRACT

The present invention provides methods for making, delivering and using formulations that combine a therapeutically active agent(s) (such as for example a Rho antagonist(s)) and a flowable carrier component capable of forming a therapeutically acceptable matrix in vivo (such as for example tissue adhesives), to injured nerves to promote repair and regeneration and regrowth of injured (mammalian) neuronal cells, e.g. for facilitating axon growth at a desired lesion site. Preferred active agents are known Rho antagonists such as for example C3, chimeric C3 proteins, etc. or substances selected from among known trans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds or Rho kinase inhibitors. The system for example may deliver an antagonist(s) in a tissue adhesive such as, for example, a fibrin glue or a collagen gel to create a delivery matrix in situ. A kit and methods of stimulating neuronal regeneration are also included.

[0001] The present invention provides methods for making, delivering andusing formulations that combine a therapeutically active agent(s) (suchas for example a Rho antagonist(s)) and a flowable carrier componentcapable of forming a therapeutically acceptable matrix in vivo (such asfor example tissue adhesives), to injured nerves to promote repair andregeneration and regrowth of injured mammalian neuronal cells, e.g. forfacilitating axon growth at a desired lesion site active agents areknown Rho antagonists such as for example C3, chimeric C3 proteins, etc.(see blow) or substances selected from among knowntrans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds (also seebelow) or Rho kinase inhibitors. The system for example may deliver anantagonist(s) in a tissue adhesive such as for example, a fibrin glue ora collagen gel to create a delivery matrix in situ. A kit and methods ofstimulating neuronal regeneration are also included.

FIELD OF THE INVENTION

[0002] The present invention pertains to the field of mammalian nervoussystem repair (e.g. repair of a central nervous system (CNS) lesion siteor a peripheral nervous system (PNS) lesion site), axon regeneration andaxon sprouting. The present invention in particular relates to a methodof delivery of C3 or other Rho antagonists to repair damage in thenervous system.

[0003] The invention also pertains to use of the delivery system fortoxicity testing of compounds applied to the injured CNS. (i.e. to acentral nervous system (CNS) lesion site or a peripheral nervous system(PNS) lesion site).

[0004] In the following by way of example only reference will generallybe made to axon growth at a a central nervous system (CNS) lesion site.

BACKGROUND

[0005] Traumatic injury of the spinal cord results in permanentfunctional impairment. Most of the deficits associated with spinal cordinjury result from the loss of axons that are damaged in the centralnervous system (CNS). Similarly, other diseases of the CNS areassociated with axonal loss and retraction, such as stroke, HIVdementia, prion diseases, Parkinson's disease, Alzheimer's disease,multiple sclerosis and glaucoma. Common to all of these diseases is theloss of axonal connections with their targets, and the ability tostimulate growth of axons from the affected or diseased neuronalpopulation would improve recovery of lost neurological functions. Forexample, following a white matter stroke, axons are damaged and lost,even though the neuronal cell bodies are alive. Treatments that areeffective in eliciting sprouting from injured axons are equallyeffective in treating some types of stroke (Boston life sciences, Sep.6, 2000 Press release). Similarly, although the the following discussionwill generally relate to delivery of Rho antagonists, etc. to atraumatically damaged nervous system, this invention also pertains todamage from unknown causes, such as during multiple sclerosis, HIVdementia, Parkinson's disease, Alzheimer's disease, prion diseases orother diseases of the CNS were axons are damaged in the CNS environment.

[0006] It has been proposed to use various agents to stimulateregeneration of cut axons, i.e. nerve lesions. Please see for examplecanadian Patent application nos. 2,304,981 (McKerracher et al) and2,300,878 (Strittmatter). These document propose the use of known Rhoantagonists such as for example C3, chimeric C3 proteins, etc. (seeblow) as well as substances selected from among knowntrans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compounds (also seebelow) or Rho kinase inhibitors for use in the regeneration of axons.

[0007] Several major advances in our understanding of axon regenerationhave led to the ability to stimulate some axon regeneration andfunctional repair in animal models of spinal cord injury. In the 1980'sexperiments by Aguayo and colleagues to use peripheral nerve grafts thatwere inserted into the brain or spinal cord showed that CNS neurons havethe capacity to regrow, and these studies highlighted that diverseclasses of CNS neurons have the potential to regenerate when given apermissive growth environment (Aguayo, et al. (1981)J ExpBiol.95:231-40). However, this technique cannot be used to rewire thecomplex circuitry of the CNS. Another major advance in our understandingof axon regeneration in the central nervous system was the discovery bySchwab and colleagues that the CNS environment did not simply lackgrowth promoting molecules, but that growth inhibitory molecules existedto block axon growth (Schwab, et al.(1993)Annu.Rev.Neurosci.16:565-595). Long distance regeneration in theCNS by blocking growth inhibitory molecules with antibodies was firstachieved in juvenile rats by neutralization of inhibitory proteinactivity with the IN-1 antibody in spinal cord (Schnell and Schwab(1990)Nature.343:269-272) and optic nerve (Weibel, et al. (1994)BrainRes.642:259-266). However, this technique suffers from the problem thatonly a single growth inhibitory protein is targeted, and delivery by theapplication of hybridoma cells or by infusing antibodies with pumps.There have been investigations on the use of growth factors to promoteregeneration in the CNS, some with notable success (Ramer, et al.(2000)Nature.403:312-316, Liu, et al. (1999)J Neurosci.19:4370-87,Blesch, et al. (1999)J Neurosci.19:3556-66). Typically infusion pumps orgene therapy techniques are used to deliver growth factors to injuredneurons. In general, trophic factors do not stimulate long distanceregeneration, but stimulate more of a local sprouting response (Schnell,et al. (1994)Nature.367: 170-173, Mansour-Robaey, et al.(1994)Proc.Natl.Acad.Sci.91:1632-1636).

[0008] A more recent advance is the demonstration that increasing theintrinsic growth capacity of neurons is sufficient to allow axonregeneration in the CNS, and that neurons primed for regeneration withneurotrophins, a conditioning lesion, or treatment with Rho antagoinstshave a better chance to grow on inhibitory substrates (Neumann(1999)Neuron.23:83-91, Cai, et al. (1999)Neuron.22:89-101, Lehmann, etal. (1999)J. Neurosci.19:7537-7547). Targeting intracellular signallingmechanisms is likely to be the most efficient way to promote axonregeneration, and it has been found that Rho antagonists are able tostimulate regeneration in the optic nerve of adult rats (Lehmann et al(1999) IBID). However, preliminary experiments to apply Rho antagoniststo the injured spinal cord were not successful. It is believed that theinfused protein was not sufficiently retained at the injury site, eitherby syringe application or the use of Gelfoam. This suggested that thedelivery of compounds that act with low affinity (compared to highaffinity neurotrophins) posed unique problems in delivery. As shall bediscussed in greater detail below the present invention relates to atissue-adhesive delivery system whereby the Rho antagonist is added tothe adhesive solution before application of the solution with a syringe,and polymerization of the adhesive within the lesion cavity in the CNS.

[0009] While neurons in the peripheral nervous system regeneratenaturally, there are many techniques used to enhance and help the repairprocess. Most of these techniques are not aimed at stimulating the rateof axonal regeneration, but in helping to guide axons back towards theirtarget regions. For example, severed nerve are sewn or glued togetherwith a fibrin glue enhance the repair process. While the followingdiscussion will generally relate or be directed at repair in the CNS,the techniques described herein may be extented to use in PNS repair.Treatment with Rho antagonists in the adhesive delivery system could beused to enhance the rate of axon growth in the PNS. This is first use ofRho antagonists in the PNS.

[0010] Growth inhibitory proteins cause growth cone collapse (Li, et al.(1996) J.Neurosci.Res.46:404-414, Fan, et al. (1993)J.CellBiol.121:867-878) and it has become clear that GTPases of the Rho familythat comprise Rho, Rac and Cdc42 are intracellular regulators of growthcone collapse (Lehmann, et al. (1999)J. Neurosci.19:7537-7547, Tigyi, etal. (1996)Journal of Neurochemistry.66:537-548, Kuhn, et al. (1999) J.Neurosci.19:1965-1975, Jin and Strittmatter(1997)J.Neurosci.17:6256-6263). These small GTPases exist in inactive(GDP) and active (GTP) forms, and the cycling between active GTP-boundand inactive GDP-bound states is tightly regulated. The guaninenucleotide exchange factors (GEFs) accelerate the release of GDP,thereby facilitating GTP binding. The GTPase activating proteins (GAPs)catalyze GTP hydrolysis and conversion of the inactive form. The GDPdissociation inhibitors (GDIs) act to maintain Rho in a GDP-bound form.GEFs for Rho all have a domain homologous with the Dbl oncoprotein, andmore than 20 such proteins have been identified, including Tiam-1 whichis highly expressed in brain (Zheng and Li (1999)J. Biol.Chem.272:4671-4679, van Leeuwen, et al. (1997)J. Cell Biol.139:797-807).Once in the active form, Rho GTPases typically stimulate ser/thrkinases, such as ROK (Rho kinase), PAK (p21-activated kinase) anddownstream effectors that act on the cytoskeleton.

[0011] The Rho family members that regulate the cytoskeleton andmotility include Rho, Rac and Cdc42 (Nobes and Hall (1995)Cell 199581:53-62). Rho is an important link between signaling through integrinsand signaling cascades of trophic factors (Laudanna, et al.(1996)Science.271:981-983, Hannigan, et al. (1996)Nature.379:91-96,Kuhn, et al. (1998)J. Neurobiol.37:524-540). Cdc42 is important for theregulation of filopodia (Nobes and Hall (1995)Cell 1995.81:53-62). BothRac and Rho regulate growth cone motility and axon growth. Innon-neuronal cells a hierarchy of signaling between Rho, Rac and Cdc42exists (Hall (1996)Ann.Rev.Cell Biol.10:31-54). In neurons Rac and Rhomay have opposite effects (van Leeuwen, et al. (1997)J. CellBiol.139:797-807, Kozma, et al. (1997)Molec. Cell. Biol.17:1201-1211).Activation of Rac stimulates outgrowth of neurites from NIE-115neuroblastoma neurons whereas activation of Rho causes neuriteretraction (van Leeuwen, et al. (1997)J. Cell Biol.139:797-807,Albertinazzi, et al. (1998)J. Cell Biol.142:815-825). In PC12 cells,dominant negative Rac disrupts neurite outgrowth in response to NGF(Hutchens, et al. (1997)Molec.Biol.Cell.8:481-500, Daniels, et al.(1998)EMBO Journal.17:754-764) whereas treatment of PC12 cells withlysophosphatidic acid (LPA), a mitogenic phospholipid that activatesRho, causes neurite retraction (Tigyi, et al. (1996)Journal ofNeurochemistry.66:537-548). The p21-activated kinase(PAK) is activatedby Rac, and PAK can also induce PC12 cell neurite outgrowth (Daniels, etal. (1998) EMBO Journal.17:754-764). It has been shown that inactivationof Rho is sufficient to promote PC12 cell neurite outgrowth on growthinhibitory substrates (Lehrnann, et al. (1999)J. Neurosci.19:7537-7547).A recent study of activating and null mutations of Rho expressed in PC12cells suggests that the differentiation state is an important parameterfor the effect of Rho on neurite outgrowth, and that priming PC12 cellswith NGF can alter the responsiveness to activating and null mutations(Sebok, et al. (1999)J. Neurochem.73:949-960). This result is inagreement with the finding that priming neurons increases intracellularcAMP (Cai, et al. (1999)Neuron.22:89-101), which can in turn influencethe activation of Rho (Lang, et al. (1996)EMBO J.15:510-519, Dong, etal. (1998)J. Biol. Chem.273:22554-22562).

[0012] In primary neurons Rac and Rho regulate both dendrite and axongrowth and cone morphology and collapse. By immunocytochemistry it hasbeen demonstrated that Rho is concentrated in growth cones, and somecolocalizes at sites of point contact (Renaudin, et al. (1998)J.Neurosci. Res.55:458-471). Experiments with activating and dominantnegative mutations have demonstrated that activation of Rac is importantin maintaining a spread morphology after challenge with growth conecollapsing factors (Kuhn, et al. (1999)J. Neurosci.19:1965-1975, Jin andStrittmatter (1 997)J.Neurosci.17:6256-6263). The activation of Rhoinduces growth cone collapse, and collapse can be prevented by treatmentwith Clostridium botulinum C3 exotransferase (hereinafter simplyreferred to as C3) (Tigyi, et al. (1996)Journal ofNeurochemistry.66:537-548, Jin and Strittmatter (1997) J.Neurosci.17:6256-6263). C3 inactivates Rho by ADP-ribosylation and isfairly non-toxic to cells (Dillon and Feig (1995)Methods in Enzymology:Small GTPases and their regulators Part. B.256:174-184).

[0013] An important downstream target of activated Rho is p160ROK, a Rhokinase (Kimura and Schubert (1992)Journal of Cell Biology.116:777-783,Keino-Masu, et al. (1996) Cell.87:175-185, Matsui, et al. (1996)EMBOJ.15:2208-2216, Matsui,.et al. (1998)J. Cell Biol.140:647-657, Ishizaki(1997)FEBS Lett.404:118-124). Among other effects, ROK phosphorylatesmyosin phosphatase to regulate actin-myosin based motility (Matsui, etal. (1996)EMBO J.15:2208-2216) and regulates proteins of the ezrinfamily (Vaheri, et al. (1997)Curr. Opin. Cell Biol.9:659-666), which areconcentrated in neuronal growth cones (Goslin, et al. (1989)J. CellBiol.109:1621-1631). Activation of ROK also induces growth conecollapse, which can be prevented by the addition of the ROK inhibitorY-27632 (Hirose, et al. (1998)J. Cell Biol.141:1625-1636).

[0014] The above studies showed that Rho antagonists can stimulateregeneration in the CNS. It has been demonstrated that Rho kinase is animportant downstream target of Rho signaling (Matsui, et al. (1996)EMBOJ.15:2208-2216, Bito (2000)Neuron.26:431-441). Among other effects,inactivation of Rho kinase stimulates neurite outgrowth in tissueculture (Bito (2000)Neuron.26:431-441) as does inactivation of Rho(Lehmann, et al. (1999)J. Neurosci.19:7537-7547). Therefore,inactivation of Rho kinase should induce the same biological effects invivo as inactivation of Rho.

[0015] The Rho kinase inhibitory Y-27632 compound is atrans-4-amino(alkyl)-1-pyridylcarbamoylcyclohexane compound; thiscompound is for example described in U.S. Pat. No. 4,997,834 the entirecontents of which are incorporated herein by reference; this patentrefers for example to compounds which may be selected from the groupconsisting of trans-4-aminomethyl-1-(4- pyridylcarbomoyl)cyclohexane,trans-4-aminomethyl-trans-1-methyl-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-cis-2-methyl-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1-(2-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1-(3-pyridylcarbamoyl)cyclohexane,trans-4-aminomethyl-1[(3-hydroxy-2-pyridylcarbamoyl)]cyclohexane,trans-4-aminomethyl-1-(3-methyl-4pyridylcarbamoyl)cyclohexane,4-(trans-4-aminomethylcyclohexylcarboxamido)-2,6-dimethyl-pyridine-N-oxide,trans-4-aminomethyl-1-(2-methyl-4-pyridylcarbamoyl)cyclohexane,trans-4-(2-aminoethyl)-1-(4- pyridylcarbamoyl)cyclohexane,trans-4-(1-amino-1-methylethyl)-1-(4-pyridylcarbamoyl)cyclohexane,trans-4-(1-aminopropyl)-1-(4-pyridylcarbamoyl)cyclohexane, andpharmaceutically acceptable acid addition salts thereof.

[0016] Please also see also Ishizali et al. 2000. Molecular Pharmacology57:976-983 3 which refers to Y-27632 in the dihydrochloride form as wellas to a related compound Y-30141, namely(R)-trans-4-(1-aminoethyl)-N-(1H-pyrrolo[2,3]pyridin-4-yl)cyclohexanecarboamide dihydrochloride. A patent application A Medicinescomparising Rho kinase inhibitor has been submitted-(EPO 956 865 A1).This inhibitor has not been tested for efficacy in CNS injury, nor hasthe company who patented this compound discovered how it might toapplied to a region of CNS injury in a kit form. Such a kit is providedin our invention. Please see also European Patent application no.97934756.4; PCT/JP97/02793; International publication # WO 98/06433(19.02.1998/07).

[0017] The compound Y-27632 has the following structure

[0018] The above structrure is used herein in a pharmaceuticallyaceptable salt form (e.g dihydrochloride salt).

[0019] The above mentioned related compound Y-30141 which may beexploited in accordance with the present invention has the followingstructure:

[0020] Agiain the above structrure may also be used herein in apharmaceutically aceptable salt form (e.g dihydrochloride salt).

[0021] The compound(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboamide(Y-27632) inhibits Rho kinase at sub-micromolar concentrations (Uehata,et al. (1997)Nature.389:990-994). Y-27632, made by a Yoshitoma, affectscalcium sensitization of smooth muscles to affect hypertension. It wasreported that the cellular target of Y-27632 is Rho-associated proteinkinase, p160ROCK (Uehata, et al. (1997)Nature.389:990-994, Somlyo(1997)Nature.389:908-911).

[0022] Different methods have been used for local delivery of drugs inthe CNS, however none of these methods have been developed as a kit withbiological component that have proven effective in the promotion of theregeneration of injured axons. IN-1 is an antibody that promotesregeneration in the CNS. One method of delivery is the implantation ofcells that secrete the active antibody (Schnell et al (1994) Nature367:170). The use of fibrin adhesive for the delivery of IN-1 antibodywas not found to be effective (Guest (1997)J. Neurosci. Res.50:888-905).Another method is the use of pumps to infuse and deliver continuouslyover time compounds that stimulate regeneration. (Ramer, et al. 2000,Nature. 403:312-316, Verge, et al. 1995. Journal of Neuroscience.15:2081-2096).

[0023] Fibrin adhesives per se have been used in studies of CNSregeneration. It has been used in replacement of sutures to graftperipheral nerves into the damaged CNS (Cheng, et al.(1996)Science.273:510-513). A fibrin glue has also been used for thedelivery of fibroplast growth factor (FGF) to damaged corticospinalneurons (Guest (1997)J. Neurosci. Res.50:888-905). The use of fibringlue plus FGF did not promote long distance regeneration.

[0024] Collagen per se has been tested for its ability to promoteregeneration after injury (Joosten (1995)J. Neurosci. Res.41:481-490.).Collagen has also been used for the delivery of neurotrophins to injuredcorticospinal axons (Houweling (1998)Expt. Neurol.153:49-59). Neither ofthe conditions was able to support long distance regeneration. In tissueculture, collagen gels can maintain gradients of small moleculesimportant in axon guidance (Kennedy, et al. (1994)Cell.78:425-435).Moreover, it had been reported that collagen gels by themselves couldfoster some axon regeneration after spinal cord injury (Joosten (1995)J.Neurosci. Res.41:481-490.).

[0025] Many different protein-based tissue adhesives exist Examplesinclude collagen gels, fibrin tissue adhesives, matrigel, lamininnetworks, and adhesives based on a composition of basment membraneproteins that contain collagen: Perhaps the most popular are the fibrinadhesives.

[0026] Fibrin sealant has three basic components: fibrinogenconcentrate, calcium chloride and thrombin. Other components can beadded to affect the properties of the gel formation. Added componentsare used to modulate time it takes for the fibrin gel to form from thesoluble components, the size of the protein network that is formed, thestrength of the gel, and protease inhibitors slow down the removal ofthe gel after it is place in the body. Several different commercialpreparations are available as kits. These include Tissucol/Tisseel,(Immuno AG, Vienna, now marketed by Baxter), Beriplast P, (Hoechst, WestGermany), and Hemaseel (Hemacure Inc. Kirkland, Quebec).

[0027] To make a fibrin gel soluble thrombin and fibrinogen are mixed inthe presence of calcium chloride. When the components mix, a fibrinadhesive gels is formed because the fibrinogen molecule is cleaved bythrombin to form fibrin monomers. The fibrin monomers spontaneously willpolymerize to form a three-dimensional network of fibrin, a reactionthat mimics the final common pathway of the clotting cascade, i.e. theconversion of fibrinogen to fibrin sealant. The key to the preparationof commercial preparations is to keep the frinogen and thrombincomponents separate until use, so that the poymerization can becontrolled with the desired timing before or after application to thebody.

[0028] Today such use of fibrin as a biologic adhesive has been widelyaccepted and found application in many fields of surgery. HEMASEELJ orTisseel VH are used as an adjunct to hemostasis in surgeries involvingcardiopulmonary bypass and treatment of splenic injuries due to blunt orpenetrating trauma to the abdomen, when control of bleeding byconventional surgical techniques, including suture, ligature and cauteryis ineffective or impractical. The action of these fibrin gels is alsoused to stop bleeding in surgical procedures involving cardipulmonarybypass and repair of the spleen. Tisseel VH has also been shown to be aneffective sealant as an adjunct in the closure of colostomies.

[0029] Collagen gels have been used in tissue culture studies to maingradients of diffusible molecules. The use of collagen gels haspermitted the identification and testing of neuronal guidance factorssuch as netrins (Kennedy, et al. (1994)Cell.78:425-435). When collagenpolymerized it forms a dense protein network. Therefore, like fibrin, ithas the potential to act as a tissue adhesive. Moreover, collagen iseasy to purify in large quantities.

[0030] There are many different types of collagens, and it is a majorcomponent of basement membranes in many different body tissues. The formof collagen often used for experimental studies in rodents is type IVcollagen because it is easily purified from rat tails.

[0031] Not only is collagen a component of the basement membrane in theperipheral nervous system, but it is known that neurons expressreceptors for collagen. Receptors for collagens are receptors of theintegrin class of proteins. One important collagen receptor expressed byneurons is the alph1 beta1 receptor (McKerracher, et al. .1996.Molec.Neurobiol. 12:95-116); this receptor is involved in the promotionof neurite outgrowth. When PC12 cells, a neuronal cell line, are platedon collagen substrates in tissue culture, collagen helps promote neuritegrowth in an integrin-dependent fashion. The addition of anit-integrinantibodies block neurite ourgrowth. Therefore, the ability of collagen,by itself, has been tested for its ability to promote axon regenerationafter spinal cord injury. It was reported that collagen gels bythemselves could foster some axon regeneration after spinal cord injury(Joosten (1995)J. Neurosci. Res.41:481-490.). However, the observedgrowth was more of a sprouting response with out any long distanceregeneration past the glial scar and site of the lesion. In addition,collagen has been tested for its ability to promote regeneration afterinjury in conjunction with the delivery of neurotrophins to injuredcorticospinal axons (Houweling (1998)Expt. Neurol. 153:49-59). Thistreatment was not able to support long distance regeneration, althoughtthe treated animals had a better sprouting response than the controls.

[0032] It would be advantageous to have a means for the direct deliveryto and maintenance at a lesion site of an agent able to facilitate axongrowth at the lesion site.

SUMMARY OF THE INVENTION

[0033] As discussed herein in acccordance with the present invention atherapeutically active agent for facilitating axon growth may bedelivered (in a flowable matrix forming substance) to a (nerve) lesionsite, for example, by injection using known syringe type glue or sealantdevices modified as necessary or desired (e.g. by addition of a furthersubstance container); examples of known delivery devices, systems,mechanisms, matrix forming compositions, and teh like are shown forexample in U.S. Pat. No. 5,989,215, U.S. Pat. No. 4,978,336, U.S. Pat.No. 4,631,055, U.S. Pat. No. 4,359,049, U.S. Pat. No. 4,974,368, U.S.Pat. No. 6,121,422, U.S. Pat. No. 6,047,861, U.S. Pat. No. 6,036,955,U.S. Pat. No. 5,945,1115, U.S. Pat. No. 5,900,408 , U.S. Pat. No.6,124,273, U.S. Pat. No. 5,922,356, and in particular U.S. Pat. No.6,117,425; the entire contents of each of these patents is incorporatedherein by reference.

[0034] A sufficient amount of a therapeutically active agent forfacilitating axon growth may be dispersed in a stable flowable (known)type of (proteinaceous) matrix forming material. Once delivered to thedesired lesion site the resulting in situ or in vivo matrix (e.g. gel orcrosslinked substances) inhibits the migration or diffusion of the agentfrom the site of injection, so as to maintain the primary effect of theagent in the region of injection, i.e. in the area of the lesion. In anyevent the active agent is to be present in an amount effective tofacilitate axon growth.

[0035] A substantially uniform dispersion of the active agent may beinitially be formed so as to provide a concentrated amount of activeagent in a physiologically acceptable matrix forming material. Thematrix forming material may be comprised of any (known) individual orcombination of peptides, proteins etc. which provides for stableplacement, or combinations thereof. Of particular interest is a collagenmaterial, a fibrinogen material, or derivatives thereof; other highmolecular weight physiologically acceptable biodegradable protein matrixforming materials may if desired be used. The active agent may, forexample, be incorporated in a sufficient concentration so as to providethe desired or effect the desired sustained release.

[0036] Typically when estimating doses in different animal species, thesame weight ratio is used. It is for example possible to apply 40 ugprotein per 20 gm mouse. Therefore, we anticipate that the ideal doseshould be approximately 3 gm per 60 kg person. We expect that the dosenecessary will depend on the size of the lesion and the time ofapplication (acute or chronic) spinal cord injury. In cases of chronicinjury, there is often a necrotic center in the spinal cord, and higherdoses may be required.

[0037] The matrix forming material may be a one-component adhesive orsealant type material (e.g. collagen material); alternatively it may bea mult-component adhesive or sealant (e.g. a fibrinogen based material).The matrix may be a human protein matrix or if necessary or desired anon-human protein matix; preferably a human protein matrix.

[0038] The (proteinaceous) matrix forming material is flowable forinjection, but once in vivo it provides for stable placement, of theactive agent in the lesion area; i.e. after injection, the active agentis released into the immediate environment the matrix providing a mediumfor prolonged contact between a lesion site and the active agent (i.e.axon growth facilatator or stimulant).

[0039] The matrix forming material(s) is (are) of course to be chosen onthe basis that the materials and resultant formed matrix will be capableon the one hand of holding the active agent for release in situ and onthe other without preventing the therapeutic effect thereof, i.e. thematrix is to be therapeutically acceptable. The choice of active agentmay be determined empirically through appropriate or suitable assayskeeping in mind that the matrix etc. are to to be therapeuticallyacceptable.

[0040] The present invention in an aspect relates to a biocompatible,(supplemented tissue sealant or adhesive) composition comprising: (i) atleast one supplement selected from the group consisting oftherapeutically active agents for facilitating axon growth; and (ii) aflowable carrier component capable of forming a pharmaceutically ortherapeuticallly acceptable matrix (in vivo)—i.e. a nerve lesion site;wherein said supplement is releasable from said matrix into the adjacentexternal environment (e.g. for a sustained period of time).

[0041] The present invention in another aspect relates a method for thepreparation of a flowable biocompatible composition comprising admixing(i) at least one supplement selected from the group consisting oftherapeutically active agents for facilitating axon growth and (ii) aflowable carrier component capable of forming a therapeuticalllyacceptable matrix in vivo at a nerve lesion site; wherein saidsupplement is releasable from said matrix into the adjacent externalenvironment.

[0042] By way of example only in accordance with the present invention amethod of applying an supplemented solution of polymerizable fibrin to adesired lesion site, may comprise a) affixing a cartridge containingimmobilized thrombin to a syringe containing a solution of fibrinogen,b) contacting the solution of fibrinogen with immobilized thrombin underconditions resulting in an activated solution of polymerizable fibrin bypassing the solution of fibrinogen through the cartridge containingimmobilized thrombin, c) adding to the fibrinogen solution or to theactivated solution a supplement (i) at least one supplement selectedfrom the group consisting of therapeutically active agents forfacilitating axon growth; and c) delivering the supplemented activatedsolution of polymerizable fibrin to the desired lesion site (e.g. acentral nervous system (CNS) lesion site or a peripheral nervous system(PNS) lesion site) under conditions which result in polymerized fibrinat the lesion site having dispersed therein the supplement wherein saidsupplement is released from said fibrin matrix into the adjacentexternal environment.

[0043] In accordance with another aspect the present invention relatesto a kit comprising, in suitable container means (e.g. separate means):(a) a first pharmaceutical composition or substance comprising abiological agent capable of facilitating axon growth; and (b) a secondpharmaceutically or therapeutically acceptable component comprising asingle flowable carrier component or two or more separate componentscapable once intermingled of forming a flowable carrier component, saidflowable carrier components each being capable of forming apharmaceutically or therapeutically acceptable matrix (e.g.proteinaceous matrix, i.e. a proteinaceous glue, proteinaceous sealant,proteinaceous gel, etc.; e.g. a human derived proteinaceous matrix) invivo at a (nerve) lesion site.

[0044] In particular the present invention provides a (axon growthstimulation) kit comprising

[0045] a) a first container means (e.g. one or more separate containers)for containing a flowable carrier component(s) or two or more separatecomponents capable once intermingled of forming a flowable carriercomponent, said flowable carrier components each being capable offorming a pharmaceutically or therapeutically acceptable matrix (e.g.proteinaceous matrix, i.e. a proteinaceous glue, proteinaceous sealant,proteinaceous gel, etc.; ie.g. a human derived proteinaceous matrix) invivo at a (nerve) lesion site (e.g. a central nervous system (CNS)lesion site or a peripheral nervous system (PNS) lesion site) and

[0046] b) a second container means for containing a therapeuticallyactive agent for facilitating axon growth at the lesion site

[0047] wherein said therapeutically active agent supplement isreleasable from said in vivo matrix into the adjacent externalenvironment (e.g. for a sustained period of time). Alternatively, ifdesired or as necessary, the first and second container means may be thesame, (e.g. a container may hold collagen and C3). The kit may ifdesired or necessary additionally comprise means for dispersing (i.e.co-mingle, blend, etc.) the therapeutically active agent in saidflowable carrier component so as to form a flowable axon growthstimulation composition as well as means for delivering the flowableaxon growth stimulation composition to the lesion site (e.g. syringeneedle). The pharmaceutically acceptable matrix may as discussed hereinbe a collagen matrix or a fibrin matrix.

[0048] In accordance with the present invention the therapeuticallyactive agent for facilitating axon growth may for example be a Rhoantagonist which may be identified by an assay method comprising thefollowing steps:

[0049] a) culturing neurons on inhibitory substrate or a substrate thatincorporates a growth-inhibitory protein.

[0050] b) Exposing the cultured neuron of step a) to a candidate Rhoantagonist in an amount and for a period sufficient to permit growth ofneurites, and determining if the candidate has elicited neurite growthfrom the cultured neurons of step a), the appearance of neurites beingsuggestive or indicative of a Rho antagonist.

[0051] A compound can be confirmed as a Rho antagonist in one of thefollowing ways:

[0052] a) Cells are cultured on a growth inhibitory substrate as above,and exposed to the candidate Rho antagonist;

[0053] b) Cells of step a) are homogenized and a pull-down assay isperformed. This assay is based on the capability of GST-Rhotektin tobind to GTP-bound Rho. Recombinant GST-Rhotektin or GST rhotektinbinding domain (GST-RBD) is added to the cell homogenate made from cellscultured as ina). It has been found that inhibitory substrates activateRho, and that this activated Rho is pulled down by(GST-RBD). Rhoantagonists will block activation of Rho, and therefore, an effectiveRho antagonist will block the detection of Rho when cell are cultured asdescribed by a) above;

[0054] c) An alternate method for this pull-down assay would be to usethe GTPase activating protein, Rho-GAP as bait in the assay to pull downactivated Rho, as described (Diekmann and Hall, 1995. In Methods inEnzymology Vol. 256 part B 207-215).

[0055] Another method to confirm that a compound is a Rho antagonist isas follows: When added to living cells antagonists that inactivate Rhoby ADP-ribosylation of the effector domain can be identified bydetecting a molecular weight shift in Rho (Lehmann et al, 1999 Ibid).The molecular weight shift can be detected after treatment of cells withRho antagonist by homogenizing the cells, separating the proteins in thecellular homogenate by SDS polyacrylamide gel electrophoresis. Theproteins are transferred to nitrocellulose paper, then Rho is detectedwith Rho-specific antibodies by a Western blotting technique.

[0056] Another method to confirm that compound is a Rho-kinaseantagonist is as follows:

[0057] a) Recombinant Rho kinase tagged with myc epitope tag, or a GSTtag is expressed in Hela cells or another suitable cell type bytransfection.

[0058] b) The kinase is purified from cell homogenates byimmunoprecipation using antibodies directed against the myc tag or theGST tag.

[0059] c) The recovered immunoprecipitates from b) are incubated with[32P] ATP and histone type 2 as a substrate in the presence or absenceof the Rho kinase. In the absence of Rho kinase activity the Rho kinaseantigens is able to block the phosphorylation activity of Rho kinase(i.e. phosphorylation of hislore), and as such identified the compoundas a Rho kinase antagonist.

[0060] The present invention is in particular,concerned with a deliverysystem and kit to apply for example, known C3, chimeric C3, or Y-27632type compounds (e.g. Y-27632, Y-30141 and the like) or a Rho kinaseinhibitor to injured regions of the CNS that include injured spinal cordor brain, and regions of the CNS injured by stroke. The nature of C3 isdiscussed herein; Y-27632 is for example mentioned above.

[0061] In the context of the present invention, the ability of C3 tostimulate (axon) regeneration in vivo was examined. Thus adult rat opticnerves were crushed an C3 applied at the same time, directly at thelesion site (Lehmann, et al. (1999)J. Neurosci.19:7537-7547). It wasfound that large numbers of axons traversed the lesion to grow in thedistal optic nerve. In particular there was for example examined thedelivery of C3 to optic nerve through the use of gelfoam an Elvax, aslow release matrix (Lehmann, et al. (1999)J. Neurosci.19:7537-7547).

[0062] It has also been found that the combination of collagen gels andC3 was able to allow axons to into the site of the glial scar. Based onexperiments with fibrin glue (see below), it is believed that deliveryof C3 in collagen may be improved by the addition of protease inhibitorsto prevent lysis of the gel and C3.

[0063] However, the present invention as mentioned above is directed tothe delivery system of a therapeutically active agent (such as forexample a Rho antogonist—C3, Y-27632, etc.) in a protein matrix thatholds the active agent (e.g. Rho antagonist) at the site of application.This delivery system retains the active agent (e.g. Rho antagonist) atthe site of CNS injury, allows large doses to be given at the site ofinjury, and prevents large amounts of the active agent (e.g. Rhoantagonist) from leaking into the systemic circulation. The proteinmatrix can either be based on the fibrin, a protein of the coagulationpathway, or it can be based on collagen, a protein of the extracellularmatrix. Both proteins when applied under specific conditions formprotein networks when polymerized. These proteins can be applied insoluble form with the additional components necessary forpolymerization, together with the Rho antagonist. When the componentsare mixed immediately before use, polymerization occurs afterapplication to the body site, in our case after application to the CNS.

[0064] The present invention as mentioned above in particular relates toa kit suitable for use in the above-described method of deliveringfibrin sealant components to a wound site. The kit comprisesindividually packaged component solutions provided in separate bottlesto prevent mixing before use, and an applicator designed so as to permitmixing of the fibrinogen/Factor XIII and thrombin with C3 at the bodysite. The kit provides pre-measured amounts of the fibrinogen and factorXIII in one bottle, the thrombin in another bottle, a C3 solution inanother bottle. The contents of the bottles would be mixed in aprescribed order, as detailed in the example below. The kit can alsoinclude one or more other storage containers which are any necessaryreagents including solvents, buffers, calcium chloride, proteaseinhibitors etc. The kit could be sold as lyophilized or frozencomponents to preserve the activity of C3 or other Rho antagonist addedto the kit.

[0065] Rho antagonist delivery system may be used in conjunction celltransplantation. Many different cell transplants have been extensivelytested for their potential to promote regeneration and repair. Theseinclude, but are not restricted to, Schwann cells (Xu, et al.(1996)Exp.Neurol. 134:261-272, Guest (1 997)Exp. Neurol. 148:502-522.,Tuszynski, et al. (1998)Cell Transplant.7:187-96), fibroblasts modifiedto express trophic factors (Liu, et al. (1999)J Neurosci.19:4370-87,Blesch, et al. (1999)J Neurosci.19:3556-66, Tuszynski, et al. (1994)ExpNeurol.126:1-14, Nakahara, et al. (1996) Cell Transplant.5:191-204),fetal spinal cord transplants (Diener and Bregman (1998)J.Neurosci.18:779-793, Bregman (.1993)Exp. Neurol. 123:2-16), macrophages(Lazarov-Spiegler, et al. (1 996)FASEB.J. 110: 1296-1302), embryonicstem cells (McDonald, et al. (1999)Nat Med.5:1410-2), and olfactoryensheathing glia (Li, et al. (1997)Science.277:2000-2002, Ramon-Cueto,et al. (1998)J Neurosci. 18:3803-15, Ramon-Cueto, et al.(2000)Neuron.25:425-35).

[0066] Brief description of the figures which illustrate exampleembodiments of the present invention:

[0067]FIG. 1A is a schematic diagram of adhesive delivery system of C3applied to an injured spinal cord wherein a tissue adhesive plus Rhoantagonist (i.e. C3) is injected into the site of ninjury;

[0068]FIG. 1B is a schematic diagram of adhesive delivery system of C3applied to an injured spinal cord wherein the injection is shown asresulting in axon regeneration thrugh the supplemented adhesion matrixand into the distal spinal cord;

[0069]FIG. 2 Schematically illustrates the model used to show efficacyin vivo. A dorsal hemisection was made in adult mice. Three to fourweeks later the anterograde tracer WGA-HRP was injected into the cortexto label the neurons of the corticospinal tract. Two days later thespinal cord was removed and and HRP enzymatic activity revealed todetect the CST axons. The corticospinal tract of adult mice was lesionat the T6 level, and the fibrin glue/C3 was added at the time of lesionwith a syringe. The expresssion CST referes to cortical spinal tract.

[0070]FIG. 3 Illustrates a longitudinal section of an untreated adultmouse spinal cord 3 weeks after lesion of the corticospinal tract viewedby darkfield microscopy. The fibres were anterogradely labeled from themotor cortex and appear fluorescent. The fibres retract back from thesite of the lesion and do not regenerate with treatment.

[0071]FIG. 4A Illustrates a low magnification view of a control animaltreated with collagen gel without C3; axons retract from the site oflesion;

[0072]FIG. 4B Illustrates a higher magnification view of a spinal cordtreated with collagen gel without C3; axons do not regenerate;

[0073]FIG. 4C Illustrates a low magnification view of labelledcorticospinal axons near the lesion site after treatment with collagengel with C3 as a Rho antagonist; axons do not retract bak from thelesion site; thy extend into the region of increased cellularity whichis the scar;

[0074]FIG. 4D Illustrates a higher magification view of FIG. C showingthat treatment with Rho antagonst is a collagen gel allows some axons tosprout into the lesion site;

[0075]FIG. 5A Illustrates a low magnification view of a spinal cordfollowing treatment with fibrin adhesive with C3 as a Rho antagonist;the section is viewed by darkfield to show the anterogradley-labeledfibres that appear white;

[0076]FIG. 5B Illustrates a hgh magnification view of the lesion siteshown in FIG. 5A showing that axons grow through the scar region; thescar appears as the verticle line;

[0077]FIG. 5C Illustrates a hgh magnification view approximately 7 mmdistal o the Ision ste of the spinal cord shown in FIGS. 5A and 5B; theregenerating fibres (arrows) grow long distances;

[0078]FIG. 6A Illustrates a darkfield microscopy of a spinal cordsection after treatment with Rho antagnist C3 in a fibrin adhesiveshowing long distance regeneration; axons sprout into the white matterand cross the lesion site;

[0079]FIG. 6B Illustrates a section of the same spinal cord shown inFIG. 6A to show axons that have regenerated a distance of 10 mm from thelesion site;

[0080]FIG. 7A Illustrates an untreated mose two days after spinal cordinjury; the control mouse is mobile but uses its front paws to dragitself forward and it shows some movement of hindlimb joints;

[0081]FIG. 7B Illustrates an animal 2 days after spinal cord injury andtreatment with C3/matrix; the animal is able to walk with weight supporttwo days after treatment;.

[0082]FIG. 7C Ilustrates a comparison of fibrin, collagen, Gelfoam andElvax methods of C3 delivery on long-distance regeneration. Animals weretreated with the test delivery system without (−C3) or with (+C3) Rhoantagonist. Distance of growth of the longest axon was scored by blindexamination of at least five sections from each animal. The longestdistance of axon growth was scored. Not shown here is that the animalsthat were not treated with Rho antagonist always showed axon retractionback from the site of lesion. When axon growth was measured, thedistance was measured from from the proximal edge of the lesion site.Each point represents data from one animal (approximately 5 sections peranimal);

[0083]FIG. 8 Is Ilustrative of open field test of behavioral recovery.Mice were scored for recovery of function by the 21 point BBB open fieldtest (see experimental section). Two phase of recovery are seen. Anearly phase, observed in all mice, although the BBB score is higher inthe C3-treated mice. The later phase of recovery of coordinatedforelimb-hindlimb movement was only observed after treatment with C3.The C3-treated mice regain almost normal walking behavior; and

[0084]FIG. 9 Is a Schematic diagram of a system exploiting a kit inaccordance with the present invention.

[0085] As used herein it is to be understood that a number of wordsand/or expressions are to have the meanings as hereinafter described.

[0086] The term “fibrin glue” or “fibrin clot” is meant to include anyformulations used to make a fibrinclot: eg tisseel VH or see (Herbert(1998)J. Biomed. Mater Res.40:551-559, Cheng, et al.(1996)Science.273:510-513, Guest (1997)J. Neurosci. Res.50:888-905).Another definition is any fibrin glue composition not sold as Tisseel,but made by combining fibrinogen, thrombin calcium ions, with or withoutother components such as factor XIII or apoprotinin.

[0087] The term “Rho antagonists” includes, but is not restricted to(known) C3, including C3 chimeric proteins, Y276321, or other Rhoantagonists delivered in the delivery system.

[0088] The term “Y276321” is defined as a Rho kinase inhibitor thatstimulated neurite outgrowth through its ability to inactive the Rhosignaling pathway (Uehata, et al. (1997)Nature.389:990-994, Bito(2000)Neuron.26:431-441).

[0089] The term “nerve injury site” refers to a site of traumatic nerveinjury or nerve injury caused by disease. The nerve injury site may be asingle nerve (eg sciatic nerve) or a nerve tract comprised of manynerves (eg. damaged region of the spinal cord). The nerve injury sitemay be in the central nervous system of peripheral nervous system in anyregion needing repair. The nerve injury site may form as a result ofdamage caused by stroke. The nerve injury site may be in the brain as aresult of surgery, brain tumour removal or therapy following a cancerouslesion. The nerve injury site may result from Parkinson's disease,Alzheimer's disease, Amyotrophic lateral sclerosis, diabetes or anyother type of neurodegenerative disease.

[0090] Rho GTPases include members of the Rho, Rac and Cdc42 family ofproteins. Our invention concerns Rho family members of the Rho class.Rho proteins consist of different variants encoded by different genes.For example, PC12 cells express RhoA, RhoB and RhoC (Lehmann et al 1999IBID). To inactivate Rho proteins inside cells, Rho antagonists of theC3 family type are effective because they inactivate all forms of Rho(eg. RhoA, Rho B etc). In contrast, gene therapy techniques, such asintroduction of a domainant negative RhoA family member into a diseasedcell, will only inactivate that specific RhoA family member.

[0091] Compounds of the C3 family from closteridium botulinum inactivateRho by ADP-ribosylation.

[0092] Recombinant C3 proteins, or C3 proteins that retain theribosylation activity are also effective in our delivery system and arecovered by this invention. In addition, Rho kinase is a well-knowntarget for active Rho, and inactivating Rho kinase has the same effectas inactiving Rho, at least in terms of neurite or axon growth (Kimuraand Schubert (1992)Journal of Cell Biology.116:777-783, Keino-Masu, etal. (1996)Cell.87:175-185, Matsui, et al. (1996)EMBO J.15:2208-2216,Matsui, et al. (1998)J. Cell Biol.140:647-657, Ishizaki (1997)FEBSLett.404:118-124), the biological activity that concerns this invention.Therefore, chemical compounds such as Y-27632, any other compound arecovered by this invention as a preferred delivery in a tissue adhesivesystem. Numerous references describing C3 type compounds can be found inMethods in Enzymology, Vol. 256, Part B, Eds.: W. E. Balch, C. H. Der,and A. Hall; Academic Press, 1995, for eg. Pgs. 196-206, 207 et seq,184-189, and 174 et seq.. In any event C3 may for example be selectedfrom the group consisting of ADP-ribosyl transferase derived fromClosteridum botulinum and a recombinat ADP-ribosyl transferase.

[0093] On the other hand any compound or molecule that does not have adirect action on Rho itself but works to decrease the function of Rhosuch as anti-sense oligos to Rho, anti-Rho kinase antibodies, and thelike. Such Rho antagonists that can be delivered in a tissue adhesivesystem are also covered by our invention. The C3 polypeptides of thepresent invention include biologically active fragments and analogs ofC3; fragments encompass amino acid sequences having truncations of oneor more amino acids, wherein the truncation may originate from the aminoterminus, carboxy terminus, or from the interior of the protein. Analogsof the invention involve an insertion or a substitution of one or moreamino acids. Fragments and analogs will have the biological property ofC3 that is capable of inactivation Rho GTPases. Also encompassed by theinvention are chimeric polypeptides comprising C3 amino acid sequencesfused to heterologous amino acid sequences. Said heterologous sequencesencompass those which, when formed into a chimera with C3 retain one ormore biological or immunological properties of C3. A host celltransformed or transfected with nucleic acids encoding C3 protein or c3chimeric protein are also encompassed by the invention. Any host cellwhich produces a polypeptide having at least one of the biologicalproperties of a C3 may be used. Specific examples include bacterial,yeast, plant, insect or mammalian cells. In addition, C3 protein may beproduced in transgenic animals. Transformed or transfected host cellsand transgenic animals are obtained using materials and methods that areroutinely available to one skilled in the art. Host cells may containnucleic acid sequences having the full-length gene for C3 proteinincluding a leader sequence and a C-terminal membrane anchor sequence(see below) or, alternatively, may contain nucleic acid sequenceslacking one or both of the leader sequence and the C-terminal membraneanchor sequence. In addition, nucleic acid fragments, variants andanalogs which encode a polypeptide capable of retaining the biologicalactivity of C3 may also be resident in host expression systems.

[0094] The Rho antogaonist that is a recombinant proteins can be madeaccording to methods present in the art. The proteins of the presentinvention may be prepared from bacterial cell extracts, or through theuse of recombinant techniques. In general, C3 proteins according to theinvention can be produced by transformation (transfection, transduction,or infection) of a host cell with all or part of a C3-encoding DNAfragment in a suitable expression vehicle.

[0095] Suitable expression vehicles include: plasmids, viral particles,and phage. For insect cells, baculovirus expression vectors aresuitable. The entire expression vehicle, or a part thereof, can beintegrated into the host cell genome. In some circumstances, it isdesirable to employ an inducible expression vector.

[0096] Those skilled in the field of molecular biology will understandthat any of a wide variety of expression systems can be used to providethe recombinant protein. The precise host cell used is not critical tothe invention. The C3 protein can be produced in a prokaryotic host(e.g., E. coli or B. subtilis) or in a eukaryotic host (e.g.,Saccharomyces or Pichia; mammalian cells, e.g., COS, NIH 3T3, CHO, BHK,293, or HeLa cells; or insect cells).

[0097] Proteins and polypeptides can also be produced by plant cells.For plant cells viral expression vectors (e.g., cauliflower mosaic virusand tobacco mosaic virus) and plasmid expression vectors (e.g., Tiplasmid) are suitable. Such cells are available from a wide range ofsources (e.g., the American Type Culture Collection, Rockland, Md.). Themethods of transformation or transfection and the choice of expressionvehicle will depend on the host system selected.

[0098] The host cells harbouring the expression vehicle can be culturedin conventional nutrient media adapted as need for activation of achosen gene, repression of a chosen gene, selection of transformants, oramplification of a chosen gene. One expression system is the mouse 3T3fibroblast host cell transfected with a pMAMneo expression vector(Clontech, Palo Alto, Calif). pMAMneo provides an RSV-LTR enhancerlinked to a dexamethasone-inducible MNITV-LTR promotor, an SV40 originof replication which allows replication in mammalian systems, aselectable neomycin gene, and SV40 splicing and polyadenylation sites.DNA encoding a C3 protein would be inserted into the pMAMneo vector inan orientation designed to allow expression. The recombinant C3 proteinwould be isolated as described below. Other preferable host cells thatcan be used in conjunction with the pMAMneo expression vehicle includeCOS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61,respectively).

[0099] C3 polypeptides can be produced as fusion proteins. For example,expression vectors can be used to create lacZ fusion proteins. The pGEXvectors can be used to express foreign polypeptides as fusion proteinswith glutathione S-transferase (GST). In general, such fusion proteinsare soluble and can be easily purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety. Another stategy to make fusion proteins isto use the His tag system.

[0100] In an insect cell expression system, Autographa californicanuclear polyhedrosis virus AcNPV), which grows in Spodoptera frugiperdacells, is used as a vector to express foreign genes. A C3 codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter, e.g., the polyhedrin promoter. Successful insertion of agene encoding a C3 polypeptide or protein will result in inactivation ofthe polyhedrin gene and production of non-occluded recombinant virus(i.e., virus lacking the proteinaceous coat encoded by the polyhedringene). These recombinant viruses are then used to infect spodopterafrugiperda cells in which the inserted gene is expressed (see, Lehmannet al for an example of making recombinant MAG protein).

[0101] In mammalian host cells, a number of viral-based expressionsystems can be utilised. In cases where an adenovirus is used as anexpression vector, the C3 nucleic acid sequence can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted into the adenovirus genome by in vitro or in vivorecombination. Insertion into a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a C3 gene product in infected hosts.

[0102] Specific initiation signals may also be required for efficienttranslation of inserted nucleic acid sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire native C3 gene or cDNA, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. In othercases, exogenous translational control signals, including, perhaps, theATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators.

[0103] In addition, a host cell may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in a specific, desired fashion. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, W138, and in particular, choroid plexus cell lines.

[0104] Alternatively, a C3 protein can be produced by astably-transfected mammalian cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public;methods for constructing such cell lines are also publicly available. Inone example, cDNA encoding the C3 protein can be cloned into anexpression vector that includes the dihydrofolate reductase (DHFR) gene.Integration of the plasmid and, therefore, the C3 protein-encoding geneinto the host cell chromosome is selected for by including 0.01-300 μMmethotrexate in the cell culture medium (as described in Ausubel et al.,supra). This dominant selection can be accomplished in most cell types.

[0105] Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are known in the art; such methods generallyinvolve extended culture in medium containing gradually increasinglevels of methotrexate. DHFR-containing expression vectors commonly usedfor this purpose include pCVSEII-DHFR and pAdD26SV(A). Any of the hostcells described above or, preferably, a DHFR-deficient CHO cell ligne(e.g., CHO DHFR cells, ATCC Accession No. CRL 9096) are among the hostcells preferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

[0106] A number of other selection systems can be used, including butnot limited to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase, and adeninephosphoribosyltransferase genes can be employed in tk, hgprt, or aprtcells, respectively. In addition, gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG-418; and hygro, which confers resistance to hygromycin can be used.

[0107] Alternatively, any fusion protein can be readily purified byutilising an antibody specific for the fusion protein being expressed.For example, a system described in Janknecht et al. (1981) Proc. Natl.Acad. Sci. USA 88, 8972, allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines. In thissystem, the gene of interest is subcloned into a vaccinia recombinationplasmid such that the gene's open reading frame is translationally fusedto an amino-terminal tag consisting of six histidine residues. Extractsfrom cells infected with recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

[0108] Alternatively, C3 or a portion thereof, can be fused to animmunoglobulin Fc domain. Such a fusion protein can be readily purifiedusing a protein A column.

[0109] It is envisioned that small molecule mimetics of the abovedescribed antagonists are also encompassed by the invention.

[0110] In the following a method to identify active Rho antagonists willbe discussed.

[0111] To test Rho antagonists for activity, a tissue culture bioassaysystem was used. This bioassay is used to define acitivity of Rhoantagonists that will be effective in promoting axon regeneration inspinal cord injury, stroke or neurodegenerative disease.

[0112] Neurons do not grow neurites on inhibitory myelin substrates.When neurons are placed on inhibitory substrates in tissue culture, theyremain rounded. When an effective Rho antagonist is added, the neuronsare able to grow neurites on myelin substrates. The time that it takesfor neurons to growth neurites upon the addition of a Rho antagonist isthe same as if neurons had been plated on growth permissive substratesuch as laminin or polylysine, typically 1 to 2 days in cell culture.The results can be scored visually. If needed, a quantitative assessmentof neurite growth can be performed. This involved measuring the neuritelength in a) control cultures where neurons are plated on myelinsubstrates and left untreated b) in positive control cultures, such asneurons plated on polylysine c) or treating cultures with differentconcentrations of the test antagonist.

[0113] To test C3 in tissue culture, it has been found that the bestconcentration is 25-50 ug/ml. Thus, high concentrations of this Rhoantagonist are needed as compared to the growth factors used tostimulate neurite outgrowth. Growth factors, such as nerve growth factor(NGF) are used at concentrations of 1-100 ng/ml in tissue culture.However, growth factors are not able to overcome growth inhibition bymyelin. Our tissue culture experiments are all performed in the presenceof the growth factor BDNF for retinal ganglion cells, or NGF for PC12cells. When growth factors have been tested in vivo, typically thehighest concentrations possible are used, in the ug/ml range. Also theyare often added to the CNS with the use of pumps for prolonged delivery(eg. Ramer et al, IBID). For in vivo experiments the highestconcentrations possible was used when working with C3 stored as a frozen1 mg/ml solution. The concentration that was chosen does not prevent thefibrin matrix from polymerizing.

[0114] For test purposes it was decided to dilute a 1 mg/ml solution ofC3 with ⅓ volume thrombin and ⅓ volume fibrinogen solutions (containcalcium and aprotinin). In order to increase the concentration of C3, itwould be possible to lyophylize C3 and then resuspend it in thefibrinogen solution. Lyophilized C3 has been tested and found to beactive. The Rho antagonist C3 is stable at 37 C for at least 24 hours.The stability of C3 was tested in tissue culture with the followingexperiment. The C3 was diluted in tissue culture medium, left in theincubator at 37 C. for 24 hours, then added to the bioassay systemdescribed above, using retinal ganglion cells as the test cell type.These cells were able to extend neurites on inhibitory substrates whentreated with C3 stored for 24 hours at 37 C. Therefore, the minimunstability is 24 hours. This is in keeping with the stability projectionsbased on amino acid composition (see sequence data, below).

[0115] In the following various tissue Adhesives and Formulations usedto make them will be discussed.

[0116] Different types of tissue adhesive can be made. Examples includecollagen gels, fibrin tissue adhesives. Other examples are matrigel,laminin networks, and adhesives based on a composition of basmentmembrane proteins that contain collagen.

[0117] Fibrin sealant has three basic components: fibrinogenconcentrate, calcium chloride and thrombin. Other components can beadded to affect the time of clot formation, and the size of the proteinnetwork that is formed. Generally when the components mix, a fibrincoagulum is formed in that the fibrinogen molecule is cleaved throughthe action of thrombin to form fibrin monomers which spontaneously willpolymerize to form a three-dimensional network of fibrin, largely kepttogether by hydrogen bonding. This corresponds to the last phase of thenatural blood clotting cascade, the coagulation rate being dependent onthe concentration of thrombin used. In order to improve the tensilestrength, covalent crosslinking between the fibrin chains is providedfor by including Factor II in the sealant composition. In the presenceof calcium ions, thrombin activates factor XIII to factor XIIIa.Activated factor XIIIa together with thrombin catalyzes thecross-linkage of fibrin and increases the strength of the clot. Thestrength of the fibrin clot is further improved by the addition offibronectin to the composition, the fibronectin being crosslinked andbound to the fibrin network formed. During wound healing the clotmaterial undergoes gradual lysis and is completely absorbed. To preventa too early degradation of the fibrin clot by fibrinolys, the fibrinsealant composition may comprise a plasminogen activator inhibitor or aplasmin inhibitor, such as aprotinin. Such an inhibitor will also reducethe fibrinolytic activity resulting from any residual plasminogen in thefibrinogen composition. Similarly, compositions may include hyaluronicacid (or other polysaccharides), and these may also comprise ahyaluronidase inhibitor such as one or more flavonoids (or correspondinginhibitors for other polysaccharides) in order to prevent degradation(i.e. to prolong the duration) of the hyaluronic acid component byhyaluronidase which is always present in the surrounding tissues. Thehyaluronic acid may, as mentioned above, be crosslinked, a commerciallyavailable example being Hylan.RTM. (trademark, available from Biomatrix,Ritchfield, N.Y., USA). The hyaluronic acid compositions may e.g. havethe form of gels, solutions, etc.

[0118] Fibrin clots in any one of the above described embodiments, maybe used for the application of a pharmaceutically active substance. Byincorporating a drug, such as an antibiotic, a growth factor, etc. intothe tissue adhesive it will be enclosed in the fibrin network formedupon application of the tissue adhesive. It will thereby be ensured thatthe drug is kept at the site of application while being controllablyreleased from the composition.

[0119] Fibrin sealant products prepared from human plasmafibrinogen/Factor XIII are available commercially. One product is atissue glue called Tisseel Fibrin Sealant (Baxter Hyland ImmunoCorporation). (Tissucol/Tisseel, Immuno AG, Vienna) and anotherBeriplast P, Hoechst, West Germany. A frozen formution of a fibrin gluedelivered with a 2 syringe system is Hemaseel made by Hemacure Inc.(Kirkland, Quebec).

[0120] In the following methods for making Tissue Adhesive Delivery kitswill be discussed.

[0121] In a preferred embodiment, the kit includes the solutionsprovided in separate bottles to prevent mixing before use, and anapplicator designed so as to permit mixing of the fibrinogen/Factor XIIIand thrombin with C3 at the body site. The kit would providepre-measured amounts of the fibrinogen and factor XIII in one bottle,the thrombin in another bottle, a calcium chloride solution in thirdbottle, and a C3 solution in a fourth bottle. The contents of thebottles would be mixed in a prescribed order, as detailed in the examplebelow. The kit can also include one or more other storage containerswhich are any necessary reagents including solvents, buffers, etc. Thekit could be sold as lyophilized or frozen components to preserve theactivity of C3 or other Rho antagonist added to the kit.

[0122] The applicator can, for example, take the form of a glass orplastic syringe with disposable needles. With a single syringe system,the components of the kit would be mixed immediately before applicationto the injury site.

[0123] A more elaborate system would allow two syringes to be attached,so that the mixing could take place in the syringe or a mixingcompartment of the syringe, before injection. One example of a twosyringe system is a Luer lock syringe, such as used for mixingadjuvants. For this a 3-way stopcocks, such as commercially available(Bio-Rad cat #7328103) is attached to the syringe so that the solutioncan be passed back and forth beore attaching the injection needle to thethird port of the 3-way stopcock. These are plastic, sterile, anddisposable.

[0124] Another method of application could be through the use of a clipto hold two syringes, and the clip would have a common plunger to ensurethat equal volumes of the thrombin and fibrinogen components are mixedin a chamber with the calcium chloride and C3, before being ejectedtrough the needle.

[0125] Other Ingredients for the Tissue Adhesive Rho Antagonist DeliverySystem are discussed hereinafter.

[0126] Other components can be added to the tissue adhesive to improveefficacy of the treatments. Such additions include growth factors,protease inhibitors, cytokines, anti-inflammatory compounds, celltransplant systems. Agents that prevent cell death, such as agents thataffect the apoptosis pathway could be added components to the deliverysystem.

[0127] Methods of Packaging Delivery System are discussed hereinafter.

[0128] In the preferred formulation, Rho antagonist, fibrinogen andthrombin are mixed together just before application, so thatpolymerization of the gel occurs in the injured CNS. Therefore, it isimportant that the fibrinogen and thrombin are package separately.However, the C3 can be packaged separately, or added to either thethrombin or fibrinogen bottles. In another formulation, the fibrinogen,thrombin and C3 are packaged together, but help at low pH, whichprevents polymerization of the gel. Polymerization would be induced bymixing this formution with a basic component that would neutralize thepH to induce coagulation of the adhesive. In another formultion, the Rhoantogonist could be added separately to the fibrinogen/thrombin mix inthe form of liposomes or other similar delivery system. Living cellscould that secrete C3 could be added as Rho antagonist.

[0129] A method of Applying Rho antagonist in vivo is discussedhereinafter.

[0130] Tissue adhesive formulations are typically applied to wound siteswith a syringe and needle. The shape of the need determine the type ofsurface that is formed when the adhesive polymerizes. In some cases,adhesives can be sprayed onto the wound surface, or into the desiredregion. This invention covers all types of syringes and needles used toapply fibrin plus Rho antagonists to injured regions of the CNS. Inaddition, it covers the addition of previously polymerized tissueadhesives with C3 to the wound. For example, fibrin can be polymerizedin a teat tube, and forcepts used to remove the gel and place it in thebody cavity. Similarly, collagen can be applied by pre-polymerizationand application by using focepts to place the gel in the injured spinalcord. One example of this is more fully explained in the example sectionof this application.

[0131] Tests were done with Gelfoam(TM), a surgical collagen-basedsponge, and Elvax, a slow 25 release plastic (Lehmann et al 1999, IBID)for the ability to deliver biologically effective concentrations of C3.Neither of these two delivery systems was effective. Therefore, onlytissue adhesive formulations (i.e. the matrix forming formulationsdiscussed herein) have efficacy in the delivery of C3 to the injured CNSin vivo.

[0132] Therapeutic Applications/Medical Uses will be Discussed Below.

[0133] The tissue adhesive system for the delivery of Rho antoagonistsmay be useful in many other conditions that affect the central andperipheral nervous system. Treatments that are effective in elicitingsprouting from injured axons are equally effective in treating sometypes of stroke (Boston life sciences, Sep. 6,.2000 Press Release).Since it has been determined that it is possible to elicit sprouting(using a kit of the present invention), it is obvious that thetreatments can be extended to stroke. Similarly, although the subject ofthis invention is related to delivery of Rho antagonists to thetraumatically damaged nervous system, this invention also pertains todamage from neurodegeneration, such as during Parkinson's disease,Alzheimer's disease, prion diseases or other diseases of the CNS wereaxons are damaged in the CNS environment. In such cases, small volumesof the tissue adhesive with C3 could be injected into the affectedregion with the use of a syringe. The treatment will cause localsprouting to restore function of neurons whose axon processes hadretracted in the course of the neurodegeneration.

[0134] Testing Example Formulation(s) and Delivery System(s) will beDiscussed Below.

[0135] Tests of invention to formulation were conducted in mice afterinjury of the corticospinal tract. All mice were tested for anatomicalregeneration of lesioned axons by anterograde tracing techniques. Someof the mice were also assessed for recovery of locomotion. The detailsof these experiments are given in the experimental section, the examplesections, and the results are shown in the figures.

EXAMPLES Example 1

[0136] A Kit for a Tissue Adhesive System.

[0137] The kit contains:

[0138] 1 vial fibringen

[0139] 1 vial apropinin solution for reconsitution of fibrinogen

[0140] 1 vial thrombin

[0141] 1 vial calcium chloride solution for reconsitution of thrombin

[0142] 1 vial C3 solution

[0143] 1.1 Lyophilized fibrinogen (75 mg/ml) in glycine buffer (2 mg/mlNaCl, 4 mg/ml trisodium citrate, 15 mg/ml glycine) was reconstituted inan aprotinin solution 3000 KIU/ml and heated to 37 C. For ease ofhandling, a combined heating and stirring device was used (appropriatevials contain a maganetic stirrer. This is called solution I.

[0144] 1.2 A thrombin solution is prepared; the solution comprisinLyophilized thrombin 500 IU/ml, 2.4 mg/ml glycine, 8 mg/ml sodiumchloride. The calcium chloride solution (40 umol CaCl2) and thrombin aremixed and heated to 37 C. This is called solution II.

[0145] 1.3 A solution of C3 (1 mg/ml) is heated to 37 C

[0146] 1.4 Equal amounts of solution I, II, and III are mixed, andimmediately drawn up in a syringe, and added to the injury site wherepolymerization occurs. Thus the C3 is added as part of the fibrin gluesolution that is placed in the lesion cavity to polymerize.

[0147] A combined heating and stirring device can be used in conjunctionwith the kit. For this, small magnetic stirrers are included in each ofthe mixing vials. The vials are then placed in the combined mixing andwarming device where the magnetic stirrer keeps the solution stirredwhile the solution is warming.

[0148] Mice that received a dorsal hemisection were treated with thefibrin/C3 adhesive. In some experiments, 10 μl of 1 mg/ml C3 inphosphate buffered saline was added to the lesion site before applyingthe C3/fibrin. Behavior recovery was assessed in an open fieldenvironment as described by Beattie, Basso and Breshnahan (1995) J.Neurotrauma 12:1-20. Anatomical regeneration was assessed by anterogradelabeling of the corticospinal fibres. Three weeks to three months afterinjury, the corticospinal fibres were labeled by inject the anterogradetracer WGA-HRP into the motor cortex as described in the art (Huang(1999)Submitted.). Two days later the animals were killed, the spinalcord removed, and longitudinal sections cut and reacted for HRPenzymatic activity, as described (Huang (1999)Submitted.). The labeledfibres were observed by microscopy to extend many mm past the lesionsite (see FIGS. 5 and 6) after treatment with C3/fibrin.

Example 2

[0149] Modification of the Kit in Example 1.

[0150] The formulation given in example 1 was used with the followingmodifications. Solution II is made with the addition of recombinant C3directly to the solution II vial. In other words, solution II containsthrombin, calcium chloride and C3. Solution I is loaded in one syringe,solution II is loaded in a second syringe. A syringe with a plunger thatsimultaneously loads both solutions is used. Thus the solutions aremixed as they enter a small chamber before the needle, and thepolymerization occurs in situ in the injured region of the CNS where thesolution is applied. The system describe here is the Duploject systemfrom Baxter Phamaceuticals U.S.A..

Example 3

[0151] Modification of the Kit in Example 1.

[0152] As example 2, but the C3 solution is mixed in vial I with thefibrinogen. Vial one and vial II are heated and prepared as described inexample 1, and injected into the injured CNS with the Duploject system.

EXAMPLE 4

[0153] Collagen Gels Used a a Tissue Adhesives.

[0154] First collagen is purified. Collagen can be purified from anysource, human or mammalian. One source of collagen is the EHS tumor cellline which is passed in mice. Collagen was purified from rat tails. Thetails were soaked in 70% alcohol for about 20 minutes. The remainingsteps were performed under aseptic conditions. The tails are brokenabout 2 cm from the tip with a hemostat and the tendon is slowly pulledout and placed in a sterile dish. The tendons are cut into small piecesand soaked in acetic acid-water (1:1000) for 48 hours in the cold. 150ml of solution is used per tail. The solution is centrifuged at 15,0000rpm, 30 min. and stored in aliquots at B10C.

[0155] Collagen gels with C3 as Rho antagonist are formed in vivo asfollows. For treatment of one mouse, 40 μg of C3 was lyophilized. The C3protein was reconstituted in 10 μl of 7.5% NaHCO₃. Collagen at 0.7 mg/mlwas used, and 25 μl collagen was added to the C3 solution. A mouse thathad received a dorsal hemisection of the spinal cord was treated with 10μl of 1 mg/ml C3 in the collagen (i.e. at the lesion site). The time ittakes for the collagen to polymerize may be modified by varying theNaHCO₃ solution. Anatomical regeneration of transected cortical spinalfibres was assessed as described in the detailed description of theinvention.

Example 5

[0156] Procedure to make recombinant C3 as a Rho antoagonist.Recombinant C3 protein was made as follows. The plasmid pGEX2T-C3 codingfor the glutathione-S-transferase (GST)-C3 fusion protein was obtainedfrom N. Lamarche (McGill Univ.). Bacteria were transformed withpGEX2T-C3, allowed to grow overnight induced with IPTG, and sonicated tobreak open the cells. The recombinant protein was purified by affinitychromatography as described (Ridley and Hall (1992)Cell.70:389-399). TheGST fusion protein was cleaved by thrombin, and thrombin was removed byincubation with 100 μl of p-aminobenzamidine agarose-beads (Sigma). TheC3 solution was dialyzed against PBS, and sterilized with a 0.22 μmfilter. The C3 concentration was evaluated by protein assay (DC assay,BioRad Labs, Missassauga, Ont.) and C3 purity was controlled by SDS-PAGEanalysis.

Example 6

[0157] Testing the Fibrin-Rho-Antagonist Formulation Using the DeliverySystem

[0158] To test the tissue adhesive system a rodent model of spinal cordinjury was used. For this, Balb-c mice were anaesthetized with 0.6 ml/kghypnorm, 2.5 mg/kg diazepam and35 mg/kg ketamine. A section of thethoracic spinal cord was exposed using fine rongers to remove the bone.A dorsal hemisection was made to cut the dorsal columns at level T6. Thefibrin/C3 adhesive was injected immediately after injury. As controlanother group of animals received fibrin alone, and a third groupreceived no treatment. The following day behavioural testing began, andcontinued for three weeks. The animals were placed in an open fieldenvironment that consisted of a rubber mat approximately 4′×3′ in size.The animals were left to move randomly, the movement of the animals werevideotaped. For each test two observers scored the animals for abilityto move ankle, knee and hip joints in the early phase of recovery. Inthe intermediate phase, the ability to support weight and correctplacement of the feet was assessed (dorsal or plantar placement). In thelate phase of recovery, the animals were assessed for correct footposition, trunk stability, and foot drag. Only animals that receivedC3/fibrin reached the late phase of recovery of coordinatedforelimb-hindlimb movement. Untreated control animals did not typicallypass beyond the early phase of recovery.

[0159] Additional Experimental Activity will be Discussed Below.

[0160] Spinal Cord Injury

[0161] To study The CST was cut bilaterally by a dorsal hemisectionextending past the central canal (1 (FIG. 2) at the T6 level. Balb-cmice were anaesthetized with 0.6 ml/kg hypnorm, 2.5 mg/kg diazepam and35 mg/kg ketamine. A section of the thoracic spinal cord was exposedusing fine rongers to remove the bone, and a dorsal hemisection was madeat level T6. Fine sissors were used to cut the dorsal half of the spinalcord, and it was recut a second time with fine knife to ensure alllesions extended past the central canal. Three weeks to four weeks afterinjury, the corticospinal fibres were labeled by injection theanterograde tracer WGA-HRP into the motor cortex as into 6 sites. Forinjection into the motor cortex a pulled glass pipette was used. Twodays later the animals were perfused transcardially with saline then 4%paraformaldehyde and the spinal cords and brains were removed.

[0162] C3 toxin was delivered locally to the site of the lesion by afibrin-based tissue adhesive delivery system (FIG. 1). Recombinant C3was mixed with fibrinogen and thrombin in the presence of CaCl₂.Fibrinogen is cleaved by thrombin, and the resulting fibrin monomerspolymerize into a three-dimensional matrix. C3 was added as part of afibrin adhesive, which polymerized within about 10 seconds after beingplaced in the injured spinal cord. Anterograde tracing with WGA-HRP wasused to study anatomical regeneration past the site of lesion in threegroups of animals: animals treated with fibrin plus C3 (C3/fibrin),animals treated with fibrin alone, and animals that did not receivedtreatment after injury (see FIG. 7). With no treatment, transected CSTaxons retract back from the site of lesion from 500 um to 1 mm (FIG. 3).Animals treated with fibrin alone showed less axon retraction, andsprouting of axons was observed to extend towards the scar. Applicationof C3 to the injured spinal cord elicited an extensive sprouting of CSTaxons into the dorsal white matter, and the axons grew into the scar andand extended past past the lesion (FIG. 4). A long distance regenerationof individual CST axons and axon bundles was elicited by C3 (FIG. 5),but not in untreated or fibrin controls. This regeneration wassignificantly different from any growth observed following treatmentwith fibrin alone.

[0163] Several different tissue adhesive delivery systems were tested.When C3 was delivered in collagen gels less axon retraction wasobserved, but the same extent of axon regeneration was not observed aswith fibrin. Gelfoam(TM), a surgical collagen sponge, was also tested.Gelfoam was not as effective as fibrin as promoting long-distanceregeneration (FIG. 7). A non-biological material, Elvax, was also testedwhich is a polymer-based artificial release system (see Lehmann et al,1999 IBD). This system was not effective in allowing cut axons access toC3.

[0164] To test functional recovery following treatment of injured spinalcord with C3, three groups of animals were score for locomotor behaviourin an open field environment according to the 21 point BBB scale (Bassoet al. ). The animals were examined by two reviewers and were placedalone in an open field environment that consisted of a rubber matapproximately 4′×3′ in size. Each animal was videotaped for approx. 3min. For the early and intermediate phases, the BBB scores were derivedfollowing observation, and confirmed by video analysis. In the latephase of recovery, the BBB score was determined from the videosprojected on a computer at 3 speed from sequences of 4 steps or more.The BBB test includes three phases of recovery: an early phase (scores1-7) of joint movement, an intermediate phase (score 8-13) where weightsupport and foot placement (dorsal or plantar) are assessed, and a latephase of coordinated movements (scores 14-21) where correct footposition, and foot drag are examined. The C3 treated animals rapidlyregained the ability to support weight (FIG. 7B) while control animalsmoved mostly by the action of their forelimbs (FIG. 7A). The controlgroups entered the intermediate recovery phase with the ability tosupport weight within one weeks, at which point they obtained theirrecovery plateau. Animals that received C3 treatment continues torecover over the 1 month period of observation, and recoveredcoordinated movement and almost normal stepping (FIG. 8).

[0165] In rats that receive a contusion injury the recovery perioddepends on the severity and location of the lesion. Typically, ratsreach a plateau of recovery by about two week, whereas after dorsalhemisection in mice it was found that the plateau of recovery is reachedwithin about 1 week. The remarkable improvement in C3-treated micewithin one day of spinal cord lesion is likely due to changes in thelocal spinal cord circuitry. These local changes might result from therobust sprouting immediately after application of C3 is applied to thetransected axons. Rates of axon growth in vivo are known to beapproximately the same as the slow axonal transport rate of 50-200um/hr. It is also possible that the local effects on the spinal cord aremechanistically different by acting on central pattern generatorsimplicated in walking behaviors or by neuroprotection immediatley aftertreatment. Most importantly, treated mice performed better immediatelyafter lesion, and they recovered almost normal walking patterns by onemonth (FIG. 8). This slower phase of recovery is attributed to thelong-distance regeneration of axons that was induced by C3 (FIG. 4).Moreover, while we only flowed the CST axons in this study, ourtreatments also are likely stimulate growth from other transected axonalpopulations.

[0166] In the following Production of recombinant C3 will be discussed.

[0167] C3 is a protein product made by the bacteria Clostridiumbotulinum. The fragment containing the C3 gene was cloned into a pGEXvector (from Amersham Pharmacia Biotech inc. Baie D'rfe, Quebec,Canada), now referred to as pGEX2T-C3, and this vetor was obtained fromNathalie Lamarche of McGill University. To confirm the C3 sequencecorresponded to that reported in the literature the insert was sequenced(see sequence below). The C3—containing pGEX vector was transformed intothe RR1 strain of E. coli (GIBCO).

[0168] Bacteria were grown in L-Broth (10 g/L Bacto-Tryptone, 5 g/LYeast Extract, 10 g/L NaCl (Fisher Scientific) withAmpicillin(BMC-Roche) at 50 ug/ml in a shaking incubator for 1 hr at 37°C. Isopropyl β-D-thiogalactopyranoside(IPTG), ( GIBCO) was added to afinal concentration of 0.5 mM to induce production of recombinantprotein and the culture was grown for a further 6 hrs at 37 C. Bacterialpellets were obtained by centrifugation, in 250 ml centrifuge bottles,at 6000 rpm at 4° C. for 5 min. Pellets can be kept frozen at −80° C. atthis time.

[0169] 5 mls of Buffer A(50 mM Tris, pH7.5, 50 mM NaCl, 5 mM MgCl2, 1 mMDTT)+1 mM PMSF was added to each pellet. Pellets were resuspended andtransferred to a 50 ml plastic beaker on ice, and a further 5 mls ofbuffer A was used to wash the centrifuge bottles. Total volume of bufferA+pellets from a 2 L culture is usually 30-40 mls. The pellets, on ice,were sonicated 5×30 secs using a BRANSON SONIFIER 450 probe sonicator.Bacteria were cooled on ice 1 minute between sonications. The sonicatewas centrifuged in a Sorvall SS-34 rotor at 10,000 rpm for 10 min at 4C. to clarify supernatant.

[0170] Glutathione-agarose beads (SIGMA#G-4510) were purchased as alyophilized powder and the beads were swollen in deionized water, thenstored in 1M NaCl at 4° C. Five ml of the beads (50% v/v) were washed ina 50 ml tube filled with buffer A (no PMSF). Tube was centrifuged at2000 rpm (500 g) for 5 min, water was removed, and replaced with bufferA. These beads were added to the cleared bacterial supernatant, andmixed for 1-2 hrs at 4 C. The beads were washed 4 times with buffer B(buffer A, NaCl is 150 mM, no PMSF), then 2× with buffer C (buffer B+2.5mM CaCl2). Washes were poured out and the beads retained each time.Next, 5 mls of thrombin(Bovine, Plasminogen-free, CALBIOCHEM #605160) 20U at (50% v/v) was added to the beads to cleave the C3 from the GSTaffinity purification tag (see cleavage site in the nucleotide sequncegiven below). This reaction was left overnight, with mixing, at 4° C.

[0171] The beads are loaded into an empty 10 ml column, PBS (phosphatebuffered saline) was added to the column and 20 l ml aliquots werecollected. To determine the location of the protein peak, 0.5 μl spotswere put on a nitrocellulose sheet, from each aliquot, and this isstained with Amido Black(Bio-rad) as a protein dot-blot. Aliquotscontaining C3 were pooled and 20 uls p-Aminobenzamidine (SIGMA#A7155)was added. The solution was mixed for 30 min at 4 C. to remove thethrombin. The C3 is centrifuged to remove the p-aminobenzamidine, andthen concentrated using a CENTRIPREP-10 concentrator (AMICON). Theconcentrated C3 is then passed through a PD-10 column (PHARMACIA,containing Sephadex G-25M) and 10 0.5 ml aliquots are collected. Adot-blot is done on these aliquots, and the appropriate aliquots(usually 3 tubes) are pooled (total volume about 1.5 mls). The purifiedrecombinant protein was filter-sterilized, aliquoted, and stored at 80°C. A protein assay was done on a small amount to determine precisely theconcentration. Purity of the recombinant C3 was evaluated by SDSpolyacrylamide gel electrophoresis. Bioactivity was assed with abioassay using either retinal ganglion cells or PC12 cells (seeidentification of Rho antagonist section).

[0172] SEQUENCE of (known) Rho Antagonist C3 Used in the Experiments

[0173] Nucleotide sequence including part of the plasmid GST sequence.The vector with the GST sequence is commercially available and thus theentire GST sequence including the start was not sequenced. It wasdesired to determine only the sequence 3′ to the thrombin cleavage sitewhich releases C3 from the GST sequence. The thrombine cleavage site isshown with an arrow and is located just to the left of the underlinednucleotide sequence below (i.e. the arrow shows the thrombin cleavagesite). The underlined sequence shows additional coding sequencetranslated in our recombinant protein that is not reported in theliterature.

[0174] Both strands were sequenced to verify that there were no errorsin the sequence.                                                    ↓ 5′GTG GCG ACC CTT CCC AAA TCG GAT CTG GTT CCG CGT GGA TCC TCT AGAGTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT AAT CAA AAG GCT TAT TCA AATACT TAC CAG GAG TTT ACT AAT ATT GAT CAA GCA AAA GCT TGG GGT AAT GCT GAGTAT AAA AAG TAT GGA CTA AGC AAA TCA GAA AAA GAA GCT ATA GTA TCA TAT ACTAAA AGC GCT AGT GAA ATA AAT GGA AAG CTA AGA CAA AAT AAG GGA GTT ATC AATGGA TTT CCT TCA AAT TTA ATA AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AATAAA ATG AAG ACC CCT GAA AAT ATT ATG TTA TTT AGA GGC GAC GAG CCT GCT TATTTA GGA ACA GAA TTT CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAAACG GCT TTT GAA AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TATGGA TAT ATT AGT ACT TCA TTA ATG AAT GTT TCT CAA TTT GCA GGA AGA CCA ATTATT ACA AAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATTAGT GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CATATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA ACAATG ATG GGC ACA GCT ATC AAT CCT AAA TAA 3′

[0175] Neucleotide sequence of recombinant C3 protein: the sequencegiven below represents the entire coding sequence for the Rho antagonistused in the experments mentioned herein. It is similar to the sequenceshown above but does not include the GST portion which when the proteinis made is enzymatically removed with thrombin. 1 GGATCCTCTA GAGTCGACCTGCAGGCATGC AATGCTTATT CCATTAATCA 51 AAAGGCTTAT TCAAATACTT ACCAGGAGTTTACTAATATT GATCAAGCAA 101 AAGCTTGGGG TAATGCTCAG TATAAAAAGT ATGGACTAAGCAAATCAGAA 151 AAAGAAGCTA TAGTATCATA TACTAAAAGC GCTAGTGAAA TAAATGGAAA201 GCTAAGACAA AATAAGGGAG TTATCAATGG ATTTCCTTCA AATTTAATAA 251AACAAGTTGA ACTTTTAGAT AAATCTTTTA ATAAAATGAA GACCCCTGAA 301 AATATTATGTTATTTAGAGG CGACGACCCT GCTTATTTAG GAACAGAATT 351 TCAAAACACT CTTCTTAATTCAAATGGTAC AATTAATAAA ACGGCTTTTG 401 AAAAGGCTAA AGCTAAGTTT TTAAATAAAGATAGACTTGA ATATGGATAT 451 ATTAGTACTT CATTAATGAA TGTTTCTCAA TTTGCAGGAAGACCAATTAT 501 TACAAAATTT AAAGTAGCAA AAGGCTCAAA GGCAGGATAT ATTGACCCTA551 TTAGTGCTTT TCAGGGACAA CTTGAAATGT TGCTTCCTAG ACATAGTACT 601TATCATATAG ACGATATGAG ATTGTCTTCT GATGGTAAAC AAATAATAAT 651 TACAGCAACAATGATGGGCA CAGCTATCAA TCCTAAATAA

[0176] Amino Acid Sequence (One Letter Code)

[0177] Translation of the above sequence to show amino acid sequence.Amino acids in bold, highlight differences from published sequence(Popoffet al. (1990) Nucl. Acid. Ress. 18:1291. EMBL accession no. X511464.) The 11 N-terminal sequences are additional; there is a singleamino acid change of an alanine (hydrophobic) to glutamic acid (Q).GSSRVDLQAC NAYSINQKAY SNTYQEFTNI DQAKAWGNAQ YKKYGLSKSE KEAIVSYTKSASEINGKLRQ NKGVINGFPS NLIKQVELLD KSFNKMKTPE NIMLFXGDDP AYLGTEFQNTLLNSNGTINK TAFEKAKAKF LNXDRLEYGY ISTSLMNVSQ FAGRPIITKF KVAKGSKAGYIDPISAFQGQ LEMLLPRHST YHIDDMRLSS DGKQIIITAT MMGTAINPK

[0178] Number of amino acids: 229

[0179] Molecular weight: 25507.5

[0180] Theoretical pI: 9.43

[0181] Amino acid composition: Ala (A) 18 7.9% Arg (R) 6 2.6% Asn (N) 187.9% Asp (D) 10 4.4% Cys (C) 1 0.4% Gln (Q) 12 5.2% Glu (E) 10 4.4% Gly(G) 16 7.0% His (H) 2 0.9% Ile (I) 18 7.9% Leu (L) 17 7.4% Lys (K) 2310.0%  Met (M) 7 3.1% Phe (F) 10 4.4% Pro (P) 7 3.1% Ser (S) 20 8.7% Thr(T) 14 6.1% Trp (W) 1 0.4% Tyr (Y) 11 4.8% Val (V) 6 2.6% Asx (B) 0 0.0%Glx (Z) 0 0.0% Xaa (X) 2 0.9%

[0182] Total number of negatively charged residues (Asp+Glu): 20

[0183] Total number of positively charged residues (Arg+Lys): 29

[0184] Estimated Half-Life:

[0185] The N-terminal of the sequence considered is G (Gly).

[0186] The estimated half-life is: 30 hours (mammalian reticulocytes, invitro).

[0187] >20 hours (yeast, in vivo).

[0188] >10 hours (Escherichia coli, in vivo).

[0189] Instability Index:

[0190] The instability index (II) is computed to be 26.88

[0191] This classifies the protein as stable.

[0192] Aliphatic index: 75.07

[0193] Grand average of hydropathicity (GRAVY): −0.479

[0194] Turning now to FIG. 9, this figure illustrates in schematicfashion a system exploiting a kit of the present invention for mixingand delivering a supplemented matrix forming material. An actualapparatus may for example be of multi-cartridge syringe type as known ormodified as necessary or desired.

[0195] The kit portion of the illustrated system comprises a containermean 1 for fibrinogen material, a container means 2 for thrombinmaterial and a container means 4 for a therapeutically active agent forfacilitating axon growth (e.g. C3 or a modified or hybrid C3). Ifdesired or necessary the the kit portion may include additionalcontainers for the separate containment of other desired or necessarycomponents; as shown the system in figure includes in dotted outline anadditional container means for the flowable matrix forming part of thekit. The system also includes a mixing container 6 wherein the C 3(hybrid) is mixed with the matrix forming elements to form thesupplemented flowable matrix forming carrier. The feed line 8 isindicative of the addition of C3 to the container 8 whereas the feedligne 10 is indicative of the addition of the flowable matrix formingelements from containers 1 and 2 and which is formed from the merging offeed lines 12 and 13. The mixing in the container means 6 may beeffected or carried out in any suitable (known) fashion, (e.g. simplestirring with a magnetic stirrer. The output line 15 of the mixingcontainer is indicative of the delivery of the supplemented mixture tothe lesion site (e.g. by needle ( e.g. syringe), pipette, etc.

[0196] Although in FIG. 9 the therapeutically active agent forfacilitating axon growth (e.g. C3) is shown As being associated with aseparate container 4, if so desired or as necessary the therapeuticallyactive agent may be associated with a container holding a flowablecarrier component (e.g. a container may hold fibrinogen and C3).

1 3 1 726 DNA Clostridium botulinum 1 gtggcgaccc ttcccaaatc ggatctggttccgcgtggat cctctagagt cgacctgcag 60 gcatgcaatg cttattccat taatcaaaaggcttattcaa atacttacca ggagtttact 120 aatattgatc aagcaaaagc ttggggtaatgctcagtata aaaagtatgg actaagcaaa 180 tcagaaaaag aagctatagt atcatatactaaaagcgcta gtgaaataaa tggaaagcta 240 agacaaaata agggagttat caatggatttccttcaaatt taataaaaca agttgaactt 300 ttagataaat cttttaataa aatgaagacccctgaaaata ttatgttatt tagaggcgac 360 gaccctgctt atttaggaac agaatttcaaaacactcttc ttaattcaaa tggtacaatt 420 aataaaacgg cttttgaaaa ggctaaagctaagtttttaa ataaagatag acttgaatat 480 ggatatatta gtacttcatt aatgaatgtttctcaatttg caggaagacc aattattaca 540 aaatttaaag tagcaaaagg ctcaaaggcaggatatattg accctattag tgcttttcag 600 ggacaacttg aaatgttgct tcctagacatagtacttatc atatagacga tatgagattg 660 tcttctgatg gtaaacaaat aataattacagcaacaatga tgggcacagc tatcaatcct 720 aaataa 726 2 690 DNA Clostridiumbotulinum 2 ggatcctcta gagtcgacct gcaggcatgc aatgcttatt ccattaatcaaaaggcttat 60 tcaaatactt accaggagtt tactaatatt gatcaagcaa aagcttggggtaatgctcag 120 tataaaaagt atggactaag caaatcagaa aaagaagcta tagtatcatatactaaaagc 180 gctagtgaaa taaatggaaa gctaagacaa aataagggag ttatcaatggatttccttca 240 aatttaataa aacaagttga acttttagat aaatctttta ataaaatgaagacccctgaa 300 aatattatgt tatttagagg cgacgaccct gcttatttag gaacagaatttcaaaacact 360 cttcttaatt caaatggtac aattaataaa acggcttttg aaaaggctaaagctaagttt 420 ttaaataaag atagacttga atatggatat attagtactt cattaatgaatgtttctcaa 480 tttgcaggaa gaccaattat tacaaaattt aaagtagcaa aaggctcaaaggcaggatat 540 attgacccta ttagtgcttt tcagggacaa cttgaaatgt tgcttcctagacatagtact 600 tatcatatag acgatatgag attgtcttct gatggtaaac aaataataattacagcaaca 660 atgatgggca cagctatcaa tcctaaataa 690 3 229 PRTClostridium botulinum 3 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn AlaTyr Ser Ile Asn 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu PheThr Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys LysTyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr LysSer Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val IleAsn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu Leu Leu AspLys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn Ile Met Leu Phe ArgGly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr Glu Phe Gln Asn Thr LeuLeu Asn Ser Asn Gly Thr Ile 115 120 125 Asn Lys Thr Ala Phe Glu Lys AlaLys Ala Lys Phe Leu Asn Lys Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr IleSer Thr Ser Leu Met Asn Val Ser Gln 145 150 155 160 Phe Ala Gly Arg ProIle Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala Gly TyrIle Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 Met Leu LeuPro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205 Ser SerAsp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 AlaIle Asn Pro Lys 225

I claim:
 1. An axon growth stimulation kit comprising a first container means for containing a flowable carrier component or two or more separate components capable once intermingled of forming a flowable carrier component, said flowable carrier components each being capable of forming a therapeutically acceptable matrix in vivo at a nerve lesion site and a second container means for containing a therapeutically active agent for facilitating axon growth at the lesion site wherein said therapeutically active agent is releasable from said in vivo matrix into the adjacent external environment.
 2. An axon growth stimulation kit as defined in claim 1 comprising means for dispersing the therapeutically active agent in said flowable carrier component so as to form a flowable axon growth stimulation composition and means for deliverying the flowable axon growth stimulation composition to the lesion site.
 3. An axon growth stimulation kit as defined in claim 1 wherein said therapeutically acceptable matrix is a collagen matrix.
 4. An axon growth stimulation kit as defined in claim 1 wherein said therapeutically acceptable matrix is a fibrin matrix.
 5. A biocompatible composition comprising: (i) at least one supplement selected from the group consisting of therapeutically active agents for facilitating axon growth; and (ii) a flowable carrier component capable of forming a therapeutically acceptable matrix in vivo at a nerve lesion site; wherein said supplement is releasable from said matrix into the adjacent external environment.
 6. A biocompatible composition as defined in claim 5 wherein said therapeutically acceptable matrix is a collagen matrix.
 7. A biocompatible composition as defined in claim 5 wherein said therapeutically acceptable matrix is a fibrin matrix.
 8. A method for the preparation of a flowable biocompatible composition comprising admixing (i) at least one supplement selected from the group consisting of therapeutically active agents for facilitating axon growth and (ii) a flowable carrier component capable of forming a therapeutically acceptable matrix in vivo at a nerve lesion site; wherein said supplement is releasable from said matrix into the adjacent external environment.
 9. A method as defined in claim 8 wherein said therapeutically acceptable matrix is a collagen matrix.
 10. A method as defined in claim 8 wherein said therapeutically acceptable matrix is a fibrin matrix. 